{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/42409"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/42409","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"The localization of two epitopes recognized by the monoclonal antibody PCG-4 on toxin A of Clostridium difficile","abstract":"Clostridium difficile causes pseudomenbranous colitis (PMC) and diarrhea in humans. Toxigenic strains of C. difficile produce two toxins. Toxin A is an enterotoxin and cytotoxin, and toxin B is a potent cytotoxin. The gene encoding toxin A has been sequenced and was shown to possess a 2.5 kb region, containing 38 similar repeating amino acid sequences, at the 3' -end of the gene. This region of the toxin A gene codes for the carbohydrate-binding portion of the toxin. The monoclonal antibody PCG-4 (MAb) binds to this portion of toxin A and neutralizes its enterotoxic activity. In addition, this monoclonal antibody has been shown to immunoprecipitate toxin A, suggesting that the MAb PCG-4 is binding to two or more similar epitopes on the toxin. The goal of this research project was to identify the neutralizing epitopes recognized by the MAb PCG-4 on the surface of the toxin A. To map the epitopes bound by the MAb PCG-4, a series of overlapping deletion clones were constructed from a 4.7 kb fragment from the 3'-end of the toxin A gene. The recombinant polypeptides expressed by these clones were tested for reactivity with the MAb PCG-4. By comparing the overlapping polypeptides, defined as either PCG-4 reactive or nonreactive, I localized the PCG-4 epitope to a 44-amino acid sequence situated between the amino acid residues 2098-2141 of toxin A A similarity search of the toxin with the 44-amino acid sequence containing the PCG-4 epitope revealed the presence of two other possible PCG-4 epitopes located between the amino acid residues 2355-2398 and 2459-2502. However, subsequent cloning experiments showed that only the region located between the amino acid residues 2355-2398 contained a PCG-4 reactive epitope. The identification of two similar epitopes within the toxin's structure explains how this monoclonal antibody is able to immunoprecipitate toxin A in the absence of subunits. Furthermore, I found that small recombinant polypeptides, containing the PCG-4 epitope lost reactivity with this monoclonal antibody following denaturation, suggesting that the epitopes recognized by this monoclonal antibody are conformationally dependent.","abstract_html":"Clostridium difficile causes pseudomenbranous colitis (PMC) and diarrhea in humans. Toxigenic strains of C. difficile produce two toxins. Toxin A is an enterotoxin and cytotoxin, and toxin B is a potent cytotoxin. The gene encoding toxin A has been sequenced and was shown to possess a 2.5 kb region, containing 38 similar repeating amino acid sequences, at the 3' -end of the gene. This region of the toxin A gene codes for the carbohydrate-binding portion of the toxin. The monoclonal antibody PCG-4 (MAb) binds to this portion of toxin A and neutralizes its enterotoxic activity. In addition, this monoclonal antibody has been shown to immunoprecipitate toxin A, suggesting that the MAb PCG-4 is binding to two or more similar epitopes on the toxin. The goal of this research project was to identify the neutralizing epitopes recognized by the MAb PCG-4 on the surface of the toxin A. To map the epitopes bound by the MAb PCG-4, a series of overlapping deletion clones were constructed from a 4.7 kb fragment from the 3'-end of the toxin A gene. The recombinant polypeptides expressed by these clones were tested for reactivity with the MAb PCG-4. By comparing the overlapping polypeptides, defined as either PCG-4 reactive or nonreactive, I localized the PCG-4 epitope to a 44-amino acid sequence situated between the amino acid residues 2098-2141 of toxin A A similarity search of the toxin with the 44-amino acid sequence containing the PCG-4 epitope revealed the presence of two other possible PCG-4 epitopes located between the amino acid residues 2355-2398 and 2459-2502. However, subsequent cloning experiments showed that only the region located between the amino acid residues 2355-2398 contained a PCG-4 reactive epitope. The identification of two similar epitopes within the toxin's structure explains how this monoclonal antibody is able to immunoprecipitate toxin A in the absence of subunits. Furthermore, I found that small recombinant polypeptides, containing the PCG-4 epitope lost reactivity with this monoclonal antibody following denaturation, suggesting that the epitopes recognized by this monoclonal antibody are conformationally dependent.","abstract_has_math":false,"creators":["Frey, Steven M."],"institution":"Virginia Tech","degree_name":"Master of Science","degree_level":"masters","degree_discipline":"Microbiology","degree_department":"Microbiology","school":null,"contributors":[],"advisors":[],"committee_chairs":["Wilkins, Tracy D."],"committee_members":["Johnson, John L.","Chen, Jiann-Shin"],"year":1990,"date_issued":"1990-12-05","date_published":"1990-12-05","updated_at":"2026-07-22T22:19:04Z","subjects":[],"languages":["en"],"rights":["In Copyright"],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-05022009-040613"],"render_values":[{"text":"etd-05022009-040613","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/10919/42409","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.committeechair","label":"Committee Chair","values":["Wilkins, Tracy D."]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Johnson, John L.","Chen, Jiann-Shin"]},{"key":"dc:contributor.department","label":"Department","values":["Microbiology"]},{"key":"dc:creator","label":"Author","values":["Frey, Steven M."]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2014-03-14T21:35:20Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2014-03-14T21:35:20Z","2009-05-02"]},{"key":"dc:date.issued","label":"Date","values":["1990-12-05"]},{"key":"dc:publisher","label":"Institution","values":["Virginia Tech"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.dcmitype","label":"Dc Type Dcmitype","values":["Text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Microbiology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["masters"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Master of Science"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["Virginia Polytechnic Institute and State University"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["In Copyright"]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://rightsstatements.org/vocab/InC/1.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-05022009-040613"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/10919/42409"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Clostridium difficile causes pseudomenbranous colitis (PMC) and diarrhea in humans. Toxigenic strains of C. difficile produce two toxins. Toxin A is an enterotoxin and cytotoxin, and toxin B is a potent cytotoxin. The gene encoding toxin A has been sequenced and was shown to possess a 2.5 kb region, containing 38 similar repeating amino acid sequences, at the 3' -end of the gene. This region of the toxin A gene codes for the carbohydrate-binding portion of the toxin. The monoclonal antibody PCG-4 (MAb) binds to this portion of toxin A and neutralizes its enterotoxic activity. In addition, this monoclonal antibody has been shown to immunoprecipitate toxin A, suggesting that the MAb PCG-4 is binding to two or more similar epitopes on the toxin. The goal of this research project was to identify the neutralizing epitopes recognized by the MAb PCG-4 on the surface of the toxin A. To map the epitopes bound by the MAb PCG-4, a series of overlapping deletion clones were constructed from a 4.7 kb fragment from the 3'-end of the toxin A gene. The recombinant polypeptides expressed by these clones were tested for reactivity with the MAb PCG-4. By comparing the overlapping polypeptides, defined as either PCG-4 reactive or nonreactive, I localized the PCG-4 epitope to a 44-amino acid sequence situated between the amino acid residues 2098-2141 of toxin A A similarity search of the toxin with the 44-amino acid sequence containing the PCG-4 epitope revealed the presence of two other possible PCG-4 epitopes located between the amino acid residues 2355-2398 and 2459-2502. However, subsequent cloning experiments showed that only the region located between the amino acid residues 2355-2398 contained a PCG-4 reactive epitope. The identification of two similar epitopes within the toxin's structure explains how this monoclonal antibody is able to immunoprecipitate toxin A in the absence of subunits. Furthermore, I found that small recombinant polypeptides, containing the PCG-4 epitope lost reactivity with this monoclonal antibody following denaturation, suggesting that the epitopes recognized by this monoclonal antibody are conformationally dependent."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Master of Science"]},{"key":"dc:format.medium","label":"Dc Format Medium","values":["BTD"]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["The localization of two epitopes recognized by the monoclonal antibody PCG-4 on toxin A of Clostridium difficile"]}]}],"canonical_facts":{"dc:contributor.committeechair":["Wilkins, Tracy D."],"dc:contributor.committeemember":["Johnson, John L.","Chen, Jiann-Shin"],"dc:contributor.department":["Microbiology"],"dc:creator":["Frey, Steven M."],"dc:date.accessioned":["2014-03-14T21:35:20Z"],"dc:date.available":["2014-03-14T21:35:20Z","2009-05-02"],"dc:date.issued":["1990-12-05"],"dc:description.abstract":["Clostridium difficile causes pseudomenbranous colitis (PMC) and diarrhea in humans. Toxigenic strains of C. difficile produce two toxins. Toxin A is an enterotoxin and cytotoxin, and toxin B is a potent cytotoxin. The gene encoding toxin A has been sequenced and was shown to possess a 2.5 kb region, containing 38 similar repeating amino acid sequences, at the 3' -end of the gene. This region of the toxin A gene codes for the carbohydrate-binding portion of the toxin. The monoclonal antibody PCG-4 (MAb) binds to this portion of toxin A and neutralizes its enterotoxic activity. In addition, this monoclonal antibody has been shown to immunoprecipitate toxin A, suggesting that the MAb PCG-4 is binding to two or more similar epitopes on the toxin. The goal of this research project was to identify the neutralizing epitopes recognized by the MAb PCG-4 on the surface of the toxin A. To map the epitopes bound by the MAb PCG-4, a series of overlapping deletion clones were constructed from a 4.7 kb fragment from the 3'-end of the toxin A gene. The recombinant polypeptides expressed by these clones were tested for reactivity with the MAb PCG-4. By comparing the overlapping polypeptides, defined as either PCG-4 reactive or nonreactive, I localized the PCG-4 epitope to a 44-amino acid sequence situated between the amino acid residues 2098-2141 of toxin A A similarity search of the toxin with the 44-amino acid sequence containing the PCG-4 epitope revealed the presence of two other possible PCG-4 epitopes located between the amino acid residues 2355-2398 and 2459-2502. However, subsequent cloning experiments showed that only the region located between the amino acid residues 2355-2398 contained a PCG-4 reactive epitope. The identification of two similar epitopes within the toxin's structure explains how this monoclonal antibody is able to immunoprecipitate toxin A in the absence of subunits. Furthermore, I found that small recombinant polypeptides, containing the PCG-4 epitope lost reactivity with this monoclonal antibody following denaturation, suggesting that the epitopes recognized by this monoclonal antibody are conformationally dependent."],"dc:description.degree":["Master of Science"],"dc:format.medium":["BTD"],"dc:format.mimetype":["application/pdf"],"dc:identifier.other":["etd-05022009-040613"],"dc:identifier.uri":["http://hdl.handle.net/10919/42409"],"dc:language.iso":["en"],"dc:publisher":["Virginia Tech"],"dc:rights":["In Copyright"],"dc:rights.uri":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:title":["The localization of two epitopes recognized by the monoclonal antibody PCG-4 on toxin A of Clostridium difficile"],"dc:type":["Thesis"],"dc:type.dcmitype":["Text"],"thesis:degree_discipline":["Microbiology"],"thesis:degree_level":["masters"],"thesis:degree_name":["Master of Science"],"thesis:institution_name":["Virginia Polytechnic Institute and State University"]},"updated_at":"2026-07-22T22:19:04Z"}